WO2003072045A2 - Traitement et prevention de la progression du sida et methodes de mise en oeuvre - Google Patents

Traitement et prevention de la progression du sida et methodes de mise en oeuvre Download PDF

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WO2003072045A2
WO2003072045A2 PCT/US2003/005514 US0305514W WO03072045A2 WO 2003072045 A2 WO2003072045 A2 WO 2003072045A2 US 0305514 W US0305514 W US 0305514W WO 03072045 A2 WO03072045 A2 WO 03072045A2
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ccr5
hiv
cxcr4
cells
protein
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WO2003072045A3 (fr
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Ghalib Alkhatib
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Indiana University
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
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    • C07—ORGANIC CHEMISTRY
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/702—Specific hybridization probes for retroviruses
    • C12Q1/703—Viruses associated with AIDS
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
    • C12N2710/24141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/24143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/40—Systems of functionally co-operating vectors

Definitions

  • the present invention relates to methods and compounds for treating HIV. More specifically, the present invention relates to methods of preventing the transmission and progression of AIDS.
  • HIV-1 is the causative agent of acquired immunodeficiency syndrome (AIDS).
  • AIDS acquired immunodeficiency syndrome
  • the virus specifically targets helper T lymphocytes and cells of the monocyte- macrophage lineage through high affinity interaction with the CD4 surface antigen (reviewed in Berger, et al., Broder, et al.).
  • CD4 surface antigen
  • a major discovery in 1996 concluded that the second receptor (coreceptor) used by HIV-1 belonged to a large family of seven transmembrane G- protein coupled receptors (Feng, et al.).
  • the CXC chemokine receptor CXCR4 is the coreceptor used by X4 HIV-1 isolates (Feng, et al.) while the CC chemokine receptor CCR5 serves as the coreceptor for R5 HIV-1 strains (Doranz, et al., Choe, et al., Alkhatib, et al., Deng, et al., Dragic, et al.).
  • the membrane orientation model of the 7TM domains chemokine receptor proteins (CCR5 is an example) is known to those in the art.
  • the amino acid sequence of the translated protein predicts seven transmembrane domains, an extracellular amino terminus and three extracellular loops, an intracellular carboxy terminus, three intracellular loops, and a S-S-proposed disulfide bond.
  • the conserved serine/Threonine rich cytoplasmic tail is the proposed domain involved in G-protein-coupled signaling.
  • Chemokine receptors share an overall 30% amino acid homology. Members of the same family share a higher degree of homology.
  • CXCR4 and CCR5 the major coreceptors for TCL-tropic and M-tropic HIV-1 , and other chemokine receptors such as CCR2b (Doranz, et al.), CCR3 (Doranz, et al., Alkhatib, et al., Choe, et al.), CCR8 (Rucker, et al.), STRL33 (BONZO) (Liao, et al., Deng, et al.) BOB (GPR15) (Deng, et al.) were also found to serve as fusion cofactors for HIV-1 entry. Despite the large number of related molecules showing an ability to employ alternate coreceptors in entry and infection, the principal coreceptors remain the initially discovered CXCR4 and CCR5 molecules.
  • HIV-1 binds to CD4 via an interaction between the first domain of CD4 and a discontinuous region of the external subunit of HIV-1 envelope glycoprotein, gp120. This region is referred to as the CD4 binding site (reviewed in Moore, et al.).
  • An accepted model for viral entry states that gp120 subunit binds first to CD4 resulting in a conformational change followed by interaction with the coreceptor which leads into another conformational change that exposes the fusion peptide of gp41 initiating the process of membrane fusion (reviewed in Moore, et al.).
  • CD4-CCR5 interaction demonstrated that the concentrations of CD4 and CCR5 required for efficient R5 infection are interdependent and that the requirements for each are increased when the other component is present in a limiting amount (Platt, et al.).
  • R5 infection requires the concerted actions of multiple CCR5 molecules and provided a mathematical model where 4-6 CCR5 monomers can be involved during R5 entry (Kuhmann, et al.).
  • CXCR4 can have the same requirement to form a complex that is essential for X4 infection (Dimitrov, et al.). Although these studies suggest physical interaction between CD4- coreceptor, the optimal stoichiometry of this association remains unknown.
  • Genotypes are represented by (+) for a wild-type allele and (-) for a ⁇ 32 allele.
  • ⁇ 32 homozygotes are referred to as -/-, ⁇ 32 heterozygotes as +/-, and to those with wild type CCR5 as +/+.
  • the amino acid structure of the ⁇ 32 mutant protein has been predicted based on the transmembrane structure of the wild type CCR5 sequence.
  • the ⁇ 32 deletion is located in a region corresponding to the second extracellular loop and results in a frameshift that produces a smaller protein which lacks the last three transmembrane domains and the carboxy terminal tail involved in G-protein signaling.
  • the frameshift caused by ⁇ 32 deletion introduces 31 new amino acid residues that are not encoded by CCR5.
  • the importance of chemokine receptors in HIV-1 transmission is highlighted by the finding that individuals homozygous for a 32-base pair deletion in CCR5 ( ⁇ 32/ ⁇ 32) are resistant to HIV-1 infection.
  • the deletion resulted in a frameshift mutation that introduced 31 new amino acid residues at the carboxy terminus of ⁇ 32 that are not present in CCR5.
  • the defective coreceptor gene encodes a prematurely terminated protein that is not detected at the cell surface and therefore is not functional as a fusion coreceptor (Samson, et al., Dean, et al., Huang, et al., Zimmerman, et al., Liu, et al.).
  • Genotypic analysis of this mutation and its distribution revealed that ⁇ 32 has a high allele frequency among Caucasians but was absent in African or Asian populations (Samson, et al., Dean, et al., Zimmerman, et al., Liu, et al.).
  • the mutant allele is not associated with any obvious phenotype in uninfected homozygous individuals.
  • Heterozygotes CCR5/ ⁇ 32 are not protected against infection, but once they become infected, have a slower progression to AIDS (Samson, et al., Dean, et al., Zimmerman, et al., Liu, et al.), indicating that partial resistance can occur in the presence of a single copy of the mutant CCR5 gene.
  • ⁇ 32 protein expression was analyzed in two brothers that are homozygous for the ⁇ 32 allele; one infected and the other uninfected. The results confirmed that the protected brother expressed ⁇ 32 protein whereas the infected brother lacked such expression. The absence of ⁇ 32 protein expression in this infected homozygote implicates a critical role for the protein in resistance to HIV-1. A growing body of evidence suggests that ⁇ 32 heterozygosity is associated with reduced risk to some complications of the disease. For example, studies analyzing the effect of +/- genotypes indicated reduced prevalence of ⁇ 32 mutation in those who do develop AIDS dementia complex (ADC) (van Rij, et al.).
  • ADC AIDS dementia complex
  • HAART highly active antiretroviral therapy
  • the resistance problem is particularly challenging because of the extraordinarily high HIV-1 mutation rate, and the ability of viral variants harboring resistance mutations in both reverse transcriptase and protease to continue replicating in vivo.
  • the viral mutability provides a rationale for developing alternate treatments.
  • the cellular receptors involved in HIV-1 entry are receiving special attention, with numerous candidate inhibitors at various stages of clinical development (Eckert, D. M. &Kim, P.S. (2001) Annu. Rev. Biochem. 70:777- 810).
  • the ⁇ 32 approach can therefore be useful to develop new methods and treatments of HIV since the ⁇ 32 protein is naturally occurring and is expressed in people who resist HIV infection.
  • Such a ⁇ 32-based treatment does not have the side effects found in the art HAART treatment of HIV-1 infection since it is aimed at decreasing the co-receptor density in a manner that is targeting both CCR5 and CXCR4, the major molecules that are responsible for disease transmission and progression, respectively.
  • individuals expressing the ⁇ 32 protein are healthy and do not show any immunological disorders.
  • a method of protecting individuals from contracting HIV by administering either a vector or a vaccine containing a sequence encoding the ⁇ 32 mutant protein containing the 31 frame shift amino acids. Also provided is a method of decreasing the amount of HIV coreceptors present on the cell surface by administering a compound having a sequence encoding the ⁇ 32 mutation in a pharmaceutically acceptable carrier. Also provided is a compound for decreasing the amount of HIV co-receptors present in the cell surface, the compound having a sequence encoding the ⁇ 32 mutant protein including the frame shift amino acids in a pharmaceutically acceptable carrier. A vector containing a sequence encoding the ⁇ 32 mutant protein is also provided. An assay for testing the efficacy of HIV treatment is also provided, the assay includes a detector for detecting the presence of the ⁇ 32 mutant protein in cells.
  • Figure 1 is a diagram showing the membrane orientation model of the 7 TM domain chemokine receptor protein
  • Figure 2 is a model showing the predicted amino acid structure of the ⁇ 32 mutant protein
  • Figures 3 A and B are graphs showing the fusion specificities of human cells expressing an adenovirus encoded CCR5 ( Figure 3A) or a vaccine virus encoded CXCR4 ( Figure 3B);
  • Figures 4A-C are flow cytometry analyses of cell surface CCR5 in a presence or absence of ⁇ 32;
  • FIGS 5 A and B are graphs showing the specific down modulation of CXCR4
  • Figures 6 A and B are graphs showing that the cells co-expressing ⁇ 32 and either CCR5 or CXCR4 are resistant to R5 and X4 fusion;
  • Figures 7 A and B are graphs showing the specificity of ⁇ 32 induced inhibition of HIV-1 Env-mediated cell fusion;
  • Figures 8 A and B are graphs showing the cell specificity of the ⁇ 32 affect;
  • Figures 9 A and B show PHA plus IL-2 activation upregulates CXCR4 and CCR5;
  • Figures 10 A and B are histograms representing staining of uninfected PMBC cells;
  • Figures 1 1 A and B are graphs showing the affect of ⁇ 32 on Env-mediated cell fusion and human PMBCs;
  • Figures 12 A-C are graphs showing PMBCs from individuals with known CCR5 genotype
  • Figures 13 A and B are graphs showing the infection kinetics of -/- and +/+ PMBCs with HIV-1 IIIB(X4) and Ba-L(R5);
  • Figures 14 A-C are photographs showing immunoblot analysis of ⁇ 32 and CCR5 proteins expressed in infected 293 cells;
  • Figures 15 A and B are photographs showing the immunodetection of native ⁇ 32 protein expressed in unstimulated PMBCs of three -/- homozygous individuals;
  • Figures 16 A-C are photographs showing the expression analysis of ⁇ 32 mRNA in -/- PMBCs ( Figure 16A) and recombinant 85 infected cells ( Figure 16B) by RT-PCR;
  • Figure 17 is a graph showing the effect of recombinant ⁇ 32 protein in X4 infection
  • Figures 18 A and B are graphs showing the Phytohemagglutinin-A + IL2- activated Ficoll purified human PBMCs which were infected with either vLA-1 (Ad5/CCR5) or vLA-2(Ad5/ ⁇ 32) at 3pfu per cell for each virus for two days that were then infected with either Ba-L(R5) or IIIB(X4);
  • Figures 19 A and B are hypothetical models of the ⁇ 32 affect; and DETAILED DESCRIPTION OF THE INVENTION
  • the present invention provides a method and composition for treating and vaccinating against HIV.
  • the composition of the present invention can be used either as a treatment for an individual who has already contracted HIV or as a vaccine to prevent the transmission of HIV. Accordingly, the composition must be administered for a sufficient period of time or at a sufficient concentration to obtain the desired effect in the individual to whom the composition has been administered.
  • composition of the present invention includes a sequence encoding the ⁇ 32 mutant protein and analogues and homologues thereof and a pharmaceutically acceptable carrier.
  • the composition can be used to create a vaccine or as a gene therapy.
  • composition of the present invention can be administered in any manner known to those of skill in the art.
  • the composition is administered either orally or intramuscularly.
  • An adenovirus vector encoding the ⁇ 32 mutant protein can be used as a vaccine which is preferably administered orally.
  • An acceptable vaccine that uses the same vector used to vaccinate against smallpox can be used to vaccinate individuals.
  • vaccine as used herein, the term is intended to include, but is not limited to a treatment which prevents HIV infection in individuals who have received the treatment thereby making the individual immune against HIV.
  • a recombinant vaccinia virus encoding the ⁇ 32 mutant protein has been constructed and used to infect human peripheral blood lymphocytes. Lymphocytes expressing the encoded ⁇ 32 mutant protein showed resistance to HIV-1 entry and infection.
  • a later administration of the vaccine can be given every month to maintain sufficient expression of the encoded ⁇ 32 mutant protein.
  • the clinical condition of the individual patient, the site and method of administration, scheduling of administration, and other factors known to medical practitioners can be taken into consideration.
  • the "effective amount" for purposes herein is thus determined by such considerations as are known in the art of vaccination wherein it must be effective to provide measurable anti-virus titer in persons given the vaccine, and, in a preferred embodiment, persons who are non-responsive to a standard anti-retroviral therapy.
  • titers can be determined, as well as proliferative assays in response to viral antigen can be run as are well known in the art.
  • the desired effect of the treatment is to either prevent the transmission of HIV or to prevent the progression of HIV infection in seropositive individuals.
  • the composition accomplishes these effects by preventing effectively the expression of coreceptors (CCR5 and CXCR4) present on the cell surface responsible for transmission and progression.
  • gene therapy refers to the transfer of genetic material (e.g DNA or RNA) of interest into a host to treat or prevent a genetic or acquired disease or condition phenotype.
  • the genetic material of interest encodes a product (e.g. the ⁇ 32 mutant protein) whose production in vivo is desired.
  • the genetic material of interest can encode a hormone, receptor, enzyme, polypeptide or peptide of therapeutic value.
  • the genetic material of interest encodes a suicide gene.
  • ex vivo and (2) in vivo gene therapy Two basic approaches to gene therapy have evolved: (1) ex vivo and (2) in vivo gene therapy.
  • ex vivo gene therapy cells are removed from a patient, and while being cultured are treated in vitro.
  • a functional replacement gene is introduced into the cell via an appropriate gene delivery vehicle/method (transfection, transduction, homologous recombination, etc.) and an expression system as needed and then the modified cells are expanded in culture and returned to the host/patient.
  • These genetically reimplanted cells have been shown to express the transfected genetic material in situ.
  • target cells are not removed from the subject rather the genetic material to be transferred is introduced into the cells of the recipient organism in situ that is within the recipient.
  • the gene is repaired in situ (Culver, 1998). These genetically altered cells have been shown to express the transfected genetic material in situ.
  • the gene expression vehicle is capable of delivery/transfer of heterologous nucleic acid into a host cell.
  • the expression vehicle can include elements to control targeting, expression and transcription of the nucleic acid in a cell selective manner as is known in the art. It should be noted that often the 5'UTR and/or 3'UTR of the gene can be replaced by the 5'UTR and/or 3'UTR of the expression vehicle. Therefore as used herein the expression vehicle can, as needed, not include the 5'UTR and/or 3'UTR of the actual gene to be transferred and only include the specific amino acid coding region.
  • the expression vehicle can include a promotor for controlling transcription of the heterologous material and can be either a constitutive or inducible promotor to allow selective transcription.
  • Enhancers that can be required to obtain necessary transcription levels can optionally be included. Enhancers are generally any non- translated DNA sequence, which works contiguously with the coding sequence (in cis) to change the basal transcription level dictated by the promoter.
  • the expression vehicle can also include a selection gene as described herein below.
  • Vectors can be introduced into cells or tissues by any one of a variety of known methods within the art. Such methods can be found generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Ml (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor, Ml (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston MA (1988) and Gilboa et al (1986) and include, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors. In addition, see United States patent 4,866,042 for vectors involving the central nervous system and also United States patents 5,464,764 and 5,487,992 for positive
  • nucleic acids by infection offers several advantages over the other listed methods. Higher efficiency can be obtained due to their infectious nature.
  • viruses are very specialized and typically infect and propagate in specific cell types. Thus, their natural specificity can be used to target the vectors to specific cell types in vivo or within a tissue or mixed culture of cells. Viral vectors can also be modified with specific receptors or ligands to alter target specificity through receptor mediated events.
  • DNA viral vector for introducing and expressing recombinant sequences is the adenovirus derived vector Adenop53TK.
  • This vector expresses a herpes virus thymidine kinase (TK) gene for either positive or negative selection and an expression cassette for desired recombinant sequences.
  • TK herpes virus thymidine kinase
  • This vector can be used to infect cells that have an adenovirus receptor, which includes most cancers of epithelial origin as well as others.
  • This vector as well as others that exhibit similar desired functions can be used to treat a mixed population of cells and can include, for example, an in vitro or ex vivo culture of cells, a tissue or a human subject.
  • Additional features can be added to the vector to ensure its safety and/or enhance its therapeutic efficacy.
  • Such features include, for example, markers that can be used to negatively select against cells infected with the recombinant virus.
  • An example of such a negative selection marker is the TK gene described above that confers sensitivity to the antibiotic gancyclovir. Negative selection is therefore a means by which infection can be controlled because it provides inducible suicide through the addition of antibiotic. Such protection ensures that if, for example, mutations arise that produce altered forms of the viral vector or recombinant sequence, cellular transformation does not occur.
  • Such features include, for example, promoter and regulatory elements that are ⁇ ⁇ specific for the desired -cell type.
  • recombinant viral vectors are useful for in vivo expression of a desired nucleic acid because they offer advantages such as lateral infection and targeting specificity.
  • Lateral infection is inherent in the life cycle of, for example, retrovirus and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells. The result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles. This is in contrast to vertical-type of infection in which the infectious agent spreads only through daughter progeny.
  • Viral vectors can also be produced that are unable to spread laterally. This characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells.
  • viruses are very specialized infectious agents that have evolved, in many cases, to elude host defense mechanisms.
  • viruses infect and propagate in specific cell types.
  • the targeting specificity of viral vectors utilizes its natural specificity to specifically target predetermined cell types and thereby introduce a recombinant gene into the infected cell.
  • the vector to be used in the methods of the invention depends on desired cell type to be targeted and is known to those skilled in the art. For example, if breast cancer is to be treated then a vector specific for such epithelial cells can be used.
  • a viral vector that is specific for blood cells and their precursors, preferably for the specific type of hematopoietic cell can be used.
  • a recombinant adeno-associated (AAV) viral vector encoding the ⁇ 32 mutant protein can be constructed and used to infect hematopoietic stem cells.
  • AAV vectors are potential gene delivery viral vectors that have the advantage of being non-pathogenic to human cells. Such vectors can provide a safe delivery and integration of the ⁇ 32 gene into hematopoietic progenitor cells. When transplanted into the patient, these cells can give rise into blood lymphocytes that express the protective ⁇ 32 mutant protein.
  • the cells expressing the ⁇ 32 mutant protein are immune to infection by a wide range of HIV-1 strains including X4, R5, and X4R5.
  • Retroviral vectors can be constructed to function either as infectious particles or to undergo only a single initial round of infection.
  • the genome of the virus is modified so that it maintains all the necessary genes, regulatory ' I sequences and packaging signals to synthesize new viral proteins and RNA. Once these molecules are synthesized, the host cell packages the RNA into new viral particles, which are capable of undergoing further rounds of infection.
  • the vector's genome is also engineered to encode and express the desired recombinant gene.
  • the vector genome is usually mutated to destroy the viral packaging signal that is required to encapsulate the RNA into viral particles. Without such a signal, any particles that are formed do not contain a genome and therefore cannot proceed through subsequent rounds of infection.
  • the specific type of vector depends upon the intended application.
  • the actual vectors are also known and readily available within the art or can be constructed by one skilled in the art using well-known methodology.
  • the recombinant vector can be administered in several ways. If viral vectors are used, for example, the procedure can take advantage of their target specificity and consequently, do not have to be administered locally at the diseased site. However, local administration can provide a quicker and more effective treatment, administration can also be performed by, for example, intravenous or subcutaneous injection into the subject. Injection of the viral vectors into a spinal fluid can also be used as a mode of administration, especially in the case of neurodegenerative diseases. Following injection, the viral vectors circulate until they recognize host cells with the appropriate target specificity for infection.
  • An alternate mode of administration can be by direct inoculation locally at the site of the disease or pathological condition or by inoculation into the vascular system supplying the site with nutrients or into the spinal fluid.
  • Local administration is advantageous because there is no dilution effect and, therefore, a smaller dose is required to achieve expression in a majority of the targeted cells. Additionally, local inoculation can alleviate the targeting requirement required with other forms of administration since a vector can be used that infects all cells in the inoculated area. If expression is desired in only a specific subset of cells within the inoculated area, then promoter and regulatory elements that are specific for the desired subset can be used to accomplish this goal.
  • non-targeting vectors can be, for example, viral vectors, viral genome, plasmids, phagemids and the like.
  • Transfection vehicles such as liposomes can also be used to introduce the non-viral vectors described above into recipient cells within the inoculated area. Such transfection vehicles are known by one skilled within the art.
  • the compound of the present invention is administered and dosed in accordance with good medical practice, taking into account the clinical condition of the individual patient, the site and method of administration, scheduling of administration, patient age, sex, body weight and other factors known to medical practitioners.
  • the pharmaceutically "effective amount" for purposes herein is thus determined by such considerations as are known in the art. The amount must be effective to achieve improvement including but not limited to improved survival rate or more rapid recovery, or improvement or elimination of symptoms and other indicators as are selected as appropriate measures by those skilled in the art.
  • the doses can be single doses or multiple doses over a period of several days, but single doses are preferred.
  • the doses can be single doses or multiple doses over a period of several days.
  • the treatment generally has a length proportional to the length of the disease process and drug effectiveness and the patient species being treated.
  • the ⁇ 32 mutation is also an effective therapeutic target for the treatment of HIV-1 infection.
  • This target can be used for developing further compositions for treating HIV infection.
  • new compounds can be developed which target both CCR5 and CXCR4 coreceptors and thereby inhibiting HIV transmission and progression.
  • additional gene therapy products such as new vectors can be developed which target the major HIV coreceptors.
  • Studies analyzing the molecular effect of this mutation have largely relied on the introduction of the ⁇ 32 DNA into cells by transfection. This method results in an inefficient delivery of the ⁇ 32 into the cells and results in expression of ⁇ 32 protein in very few cells in a sample monolayer.
  • Applicants have developed adenovirus and vaccinia virus vectors that deliver the ⁇ 32 DNA efficiently into every cell in the monolayer. As a result, 100% of the cells transduced with the ⁇ 32 vector express this protein ( ⁇ 32 protein).
  • ⁇ 32 heterozygosity (individuals carrying one copy of ⁇ 32) is associated with reduced risk to some complications of the disease.
  • studies analyzing the effect of ⁇ 32 genotypes indicated reduced prevalence of ⁇ 32 mutation in those who do develop AIDS dementia complex (ADC).
  • Others reported an association of ⁇ 32 mutation with protection against HIV-associated lymphoma.
  • Recent reports described that carriers of the ⁇ 32 mutation respond better to highly active antiretroviral therapy (HAART) treatment compared to wild type controls.
  • HAART highly active antiretroviral therapy
  • CD8+ T cells purified from ⁇ 32 carriers showed increased ability to kill virally infected cells. All the above studies point to the potential use of the ⁇ 32 effect in other disease conditions.
  • HIV-1 Human immunodeficiency virus type 1
  • CD4 a coreceptor, principally the CCR5 and/or CXCR4 chemokine receptors, for entry into host cells.
  • CCR5 The central role of CCR5 in HIV-1 transmission and pathogenesis has been highlighted by the epidemiological and genetic identification of powerful disease modifying effects of the naturally occurring CCR ⁇ 32 allele, a 32 base pair deletion encoding a truncated and non-cell surface expressed version of the receptor.
  • CCR ⁇ 32 homozygotes are rarely found among HIV-1 infected individuals.
  • HIV-1 infected CCR ⁇ 32 heterozygotes progress more slowly to AIDS than individuals lacking this allele.
  • Previous studies have indicated that ⁇ 32 protein binds to wild-type CCR5 inside the cell and can retard the transport of functional CCR5 to the cell surface.
  • this mechanism did not explain why CXCR4 only rarely could compensate for the CCR5 deficiency and allow X4 or dual tropic (R5X4) virus infection.
  • the present invention shows that the ⁇ 32 protein down-regulates the major coreceptors resulting in an unfavorable of the molecules involved in membrane fusion.
  • PBMCs from ⁇ 32/ ⁇ 32 individuals express lower surface levels of CXCR4 in comparison to wild-type CCR5 PBMCs. Further, lower X4 fusion/infection activity was correlated in the ⁇ 32/ ⁇ 32 cells.
  • replication- defective adenovirus type 5 (Ad5) vectors were constructed encoding either CCR5 or ⁇ 32 proteins.
  • the Ad5 gene delivery system results in efficient delivery of the gene of interest to most cells in a monolayer resulting in sufficient expression levels to allow detailed analysis and characterization of the ⁇ 32 protein and examination of its potential role in the protective phenotype.
  • One mechanism of genetic resistance to HIV-1 is caused by the unique activity of the ⁇ 32 protein that results in an unfavorable stoichiometry of the surface proteins involved in HIV-1 entry.
  • ⁇ 32 interacts specifically with the major coreceptors CXCR4 and CCR5 and impairs the formation of coreceptor complexes and CD4-coreceptor interaction resulting in an imbalance of the optimal stoichiometry of the molecules involved in the process of membrane fusion.
  • the experimental approach described herein enables one to characterize the ⁇ 32 protein at the molecular level and determine its contribution to resistance to HIV-1 infection.
  • the observed effect of ⁇ 32 on both CCR5 and CXCR4 implicates a common interaction site for the principal coreceptors on ⁇ 32.
  • ⁇ 32 protein encoded by a recombinant vaccinia virus was also studied. This can also be in the form of an attenuated vaccinia virus that is used for smallpox vaccination.
  • a recombinant vaccinia virus that expresses the ⁇ -32 protein with expression that results in cells that are resistant to HIV-1 entry has also been established. The results of the experiment are shown in Figure XY.
  • Cells (MAGI-CCR5) were infected with recombinant vaccinia viruses encoding the proteins indicated at the X-axis. These cells were challenged with cells expressing the two major prototypic HIV-1 envelope glycoproteins (LAV or Ba-L).
  • the extent of cell fusion was determined by measuring the production of ⁇ - galactosidase. The results show that cells expressing ⁇ 32 are resistant to HIV-1 fusion.
  • Two controls were included in this experiment. The first control is the use of cells infected with the vaccinia vector (WR), the vector used to generate the ⁇ 32/vaccinia recombinant virus. The second control is the use of cells infected with a vaccinia recombinant encoding CCR5. The two controls showed no resistance to HIV-1 fusion as demonstrated in the figure.
  • ⁇ 32 protein was also investigated. Delta-32 RNA and protein expression in (-/-) PBMCs was also examined. RNA expression of ⁇ 32 was verified by RT-PCR analysis. The immunoblot analysis revealed a protein band that corresponds to the same molecular weight band seen with recombinant protein analysis. These protein bands were not obtained with the preimmune serum. The identity of the band that appears above the 34 Kd marker band is not known at present, however, since it is expressed in normal CCR5 individuals, it could represent a cross-reactive cellular protein. The analysis also revealed that the ⁇ 32 protein band detected in the protected (-/-) individuals was absent in an infected (-/-) individual suggesting a critical role for the ⁇ 32 protein in resistance to HIV-1.
  • the recombinant vaccinia virus encoding the ⁇ 32 protein can be used as a live attenuated vaccine to deliver the ⁇ 32 protein into susceptible cells.
  • the mechanism of protection obtained with this recombinant virus will be investigated in a mouse model that expresses human CXCR4, CCR5 and CD4.
  • the mode of administration can be intramuscular or intravenous.
  • the C-terminal region of the ⁇ 32 protein created novel epitopes that are not native to people who do not express ⁇ 32 protein. Therefore, antibody generation to those novel epitopes in ⁇ 32 occur in vaccinated mice. These antibodies can be examined for their ability to neutralize HIV-1 infection.
  • Adeno-associated viral vectors are also potential vectors for gene therapy (Zhou, et al., Gene Therapy, 3:223-229 (1996)). These vectors also contain the herpes virus TK promoter-driven neomycin gene for selection. AAV- based vectors have the advantage of being non-pathogenic and the fact that they integrate into the host chromosome by a site-specific manner. Therefore, they do not appear to lead to insertional mutations. HIV-based lentivirus vectors can also be utilized. Recombinant HIV-1 vector expressing the ⁇ 32 protein can be constructed and used to treat HIV-1 infection in cultured peripheral blood lymphocytes that are chronically infected with HIV-1.
  • the idea is that the recombinant ⁇ 32/HIV integrates into the host chromosome at the same sites that contain the wild type genomic copies of HIV-1. Recombination results in the insertion of the ⁇ 32 gene into the host chromosome thereby providing continuous expression of the ⁇ 32 protein.
  • This approach can be very beneficial for those patients who do not respond to HAART and are therefore left to their fate and to the virus controlling their immune systems.
  • the idea of generating HIV-based vectors to trap HIV-1 virus has been previously suggested (Endres, et al., Science, 278:1462-1464 1997). This approach offers a method for delivering the ⁇ 32 protein directly to HIV-infected cells in vivo and provides an additional treatment strategy in conjunction with existing HAART therapy.
  • PCR Polymerase chain reaction
  • Example 1 In order to study the role of ⁇ 32 in genetic resistance to HIV-1 infection, a vector system is needed that can reproducibly deliver the ⁇ 32 transcription unit into most cells in a monolayer that result in sufficient quantities of recombinant ⁇ 32 in a variety of cell types.
  • the immunological reagents must also be developed that enable one to specifically detect native and recombinant ⁇ 32 proteins.
  • a number of cDNA clones have been obtained encoding several CC and CXC chemokine receptors. Most of these receptors were subcloned under the control of the synthetic early/late promoter of vaccinia virus to produce higher levels of expression of the chemokine receptor protein.
  • the original cDNA clones were all under the T7 promoter, and are used when lower levels expression of the protein is desired.
  • a number of recombinant vaccinia viruses made by Chris Broder were obtained from the laboratory of Ed Berger, N1AID.NIH. These recombinants are used to prepare cells expressing a variety of HIV-1 envelope glycoproteins (Envs) derived from different X4, R5, or R5/X4 isolates.
  • Envs HIV-1 envelope glycoproteins
  • the immuno-detection of recombinant proteins is critical to verify expression, localization, and quantification of the molecules being tested.
  • Polyclonal antibodies against synthetic peptides corresponding to the amino termini of CXCR4 (Feng, et al.) and CCR5 (Alkhatib, et al.) have been described and are available for use in the laboratory. It was shown that these antibodies detect recombinant CXCR4 expressed by using the vaccinia virus vectors, and endogenous CXCR4 made in peripheral blood macrophages and lymphocytes.
  • the anti-CXCR4 peptide antibodies do not detect surface CXCR4.
  • monoclonal antibodies are used that have been obtained commercially.
  • ⁇ 32 a recombinant adenovirus encoding either wild type CCR5 or the deletion mutant ⁇ 32 protein, referred to as ⁇ 32 has been constructed.
  • the entire cDNA fragments encoding either ⁇ 32 or CCR5 were individually
  • Ad5/CCR5 DNA was used to isolate positive recombinant viral plaques on 293 cell monolayers. Southern blot analysis was subsequently performed to confirm the predicted structure of recombinant viruses without deletions or rearrangements, even after three rounds of plaque purification and propagation. The recombinant viruses consistently grew to titers ranging between 7x10 8 to 1x10 9 plaque forming units (PFU)/ml in 293 cells.
  • PFU plaque forming units
  • Ad5/MVHA Ad5/MVHA
  • Ad ⁇ MVPC Ad5/MVHA
  • Ad ⁇ MVPC Ad5/MVHA
  • Ad ⁇ MVPC recombinant Ad5 viruses encoding either measles virus (MV) haemaglutinin (Alkhatib, et al.) or MV phosphoprotein Alkhatib, et al.) were used as negative controls for all subsequent experiments to measure the effect of recombinant ⁇ 32 protein on cell surface expression of the major coreceptors and their fusion activities.
  • Wild type Ad5 is a replication competent virus that does not contain DNA sequences that are specific for ⁇ 32 or any chemokine receptor. Expression of MVHA or MVPC by Ad5 was verified by surface staining using MV-specific polyclonal antibodies as previously described (Alkhatib, et al.).
  • the experimental approach involves the analysis of fusion between two distinct cell populations, one expressing CD4 (endogenous or encoded by a recombinant virus) and the other expressing the indicated HIV-1 envelope glycoprotein encoded by a recombinant vaccinia virus.
  • Cell fusion is scored by a reporter gene activation assay in which the cytoplasm of one cell population expressing vaccinia virus-encoded T7 RNA polymerase and the cytoplasm of the other expressing lac Z gene linked to the T7 promoter; cell fusion activates ⁇ - Galactosidase.
  • the advantages of preparing target cells (cells expressing the coreceptor) using the Ad5 expression system include:
  • the Ad5 cytopathic effect is much less severe than vaccinia, 2) The cells are healthier and easier to manipulate; 3) The recombinant Ad5 viruses are efficient high-level expressing vectors that result in infection of all cells in a monolayer; 4) The G-protein signaling pathway is not impaired in Ad5-infected cells (Braciak, et al.) compared to vaccinia virus infected cells; 5) The recombinant Ad5 viruses are replication-defective in cells other than 293 and can be useful to analyze without the cytopathic effect associated with the wild type virus; and 6) The recombinant Ad5 viruses provide a much better signal to background ratio.
  • Co-expression of proteins is accomplished with the Ad5 vector system by infecting cells with two different recombinant viruses at the same time. This is a well established method for expressing two different proteins in a variety of cell types due to the broad host specificity of Ad5 for mammalian cells (Alkhatib, et al.).
  • the basic features of the fusion assay were developed by using the HIV-1 Env-CD4 interaction of two different populations of cells, one expressing CD4 and the other expressing the HIV-1 Env (Nussbaum, et al., Alkhatib, et al.).
  • adenovirus expression system The rationale for using an adenovirus expression system is that cells infected with this vector can, unlike vaccinia infected cells, respond to calcium flux assays making it possible to analyze the response of infected cells to different chemokines. Moreover, the recombinant Ad5 viruses used are replication-defective and do not cause severe cytopathic effect in a wide range of mammalian cells including primary cells. In the vaccinia-based cell fusion assay, CCR5 functions as a fusion coreceptor for Envs from several R5-tropic HIV-1 isolates, as well as the R5/X4 strain 89.6 (Alkhatib, et al.).
  • human 293 cells were used as the target cell population after transfection with pCDNA3/CD4 and coinfection with Ad5T7 ⁇ -gal (lacZ under T7, from Frank Graham and vLA-1 (CCR5). Hela cells coinfected with vTF7-3 (T7 RNA polymerase) and one of the HIV-1 Envs as effector cells were used. After mixing the effector and target cell populations and incubation at 37°C for 2.5 hours, fusion specificity of HIV-1 Envs was measured by ⁇ -gal production in a colorimetric lysate assay.
  • Monoclonal antibodies to CCR5 were used to detect coreceptor density at the cell surface of infected cells. Surface levels of CCR5 were significantly reduced in the presence of recombinant ⁇ 32 protein ( Figure 4), consistent with previous published observations that examined the effect of ⁇ 32 on cell surface expression of CCR5 (Benkirane, et al., Wu, et al., Paxton, et al.).
  • a human CCR5/293 cell line and another CXCR2/293 cell line were used to analyze the effect of ⁇ 32 on CXCR4.
  • Human CCR5/293 cells were infected with VLA-2 ⁇ 32), vLA-1 (CCR5), Ad ⁇ MVHA (MVHA), Ad ⁇ MVPC (MVPC), or Ad5 and stained with Mabs against CXCR4.
  • the results of this analysis demonstrate that cells expressing ⁇ 32 but not CCR5, MVHA, or MVPC showed downmodulation of CXCR4 ( Figure 5A).
  • CXCR2 is a CXC chemokine receptor that shares 35% homology with CXCR4.
  • CXCR2 or CXCR4 surface levels were not significantly altered in cells expressing either CCR5 or MVHA - ( Figure 6B, CCR5/CXCR2 and MVHA/CXCR4 line graphs) indicating the specificity of ⁇ 32 downmodulation effect.
  • EGFR epidermal growth factor receptor
  • a glycoprotein expressed on most cells including 293 cells and on CCR2 in a human CCR2/293 cell line were examined.
  • FIG. 4 shows the flow cytometry analysis of cell surface CCR5 in the presence or absence of ⁇ 32.
  • Cells (293 cells) expressing CCR5 and/or ⁇ 32 were treated with monoclonal antibodies and detected by indirect staining using fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin G. The values shown at the right corner of each histogram represent mean fluorescence intensity.
  • Figure 5 shows the specific downmodulation of CXCR4.
  • Figure 5A shows human 293 cells which were infected at 10 pfu/cell with the different Ad5 viruses encoding wild type adenovirus proteins (Ad5), measles virus hemaglutinin (MVHA), measles C& phosphoproteins (MVPC), CCR5, or ⁇ 32 and stained for surface CXCR4. Reduction in surface CXCR4 was only observed with cells expressing ⁇ 32 but not other proteins such as CCR5, MVHA, or MVPC.
  • Ad5 wild type adenovirus proteins
  • MVHA measles virus hemaglutinin
  • MVPC measles C& phosphoproteins
  • Figure 5B shows ⁇ 32-specific downmodulation of CXCR4 in a CXCR2/293 cell line.
  • Cells were infected at increasing moi's with either vLA-1 (CCR5/Ad5), Ad5/MVHA, or vLA-2 (Ad5/ ⁇ 32), then stained with monoclonal antibodies to CXCR4 or CXCR2 (R&D).
  • ⁇ 32 expression resulted in reduced surface staining of CXCR4 ( ⁇ /CXCR4) but had no significant effect on CXCR2 surface expression ( ⁇ /CXCR2).
  • Expression of MVHA, or CCR5 by the adenovirus system had no downmodulation effect on cell surface expression of CXCR4 or CXCR2.
  • a human 293/CD4 Cell line was generated and cells were infected with either vLA-1 (CCR5), vLA-2 ⁇ 32), Ad5, or coinfected with vLA-1+vLA-2 (CCR5+ ⁇ 32) then infected with Ad5Pol3 (T7 RNA polymerase).
  • vCB-21 R and either vCB-16 (Unc), . vCB-41 (LAV), or vCB-39 (ADA). Fusion activity was scored by the amount of ⁇ -galactosidase produced.
  • Figure 6 shows cells coexpressing ⁇ 32 and either CCR5 or CXCR4 are resistant to R5 and X4 fusion.
  • Human 293 cells expressing CCR5 and CXCR4 (endogenous), or coexpressing CCR5+ ⁇ 32 or ⁇ 32 were challenged with effector Hela cells expressing the indicated HIV-1 Envs. Cell fusion was scored by ⁇ -gal production. This analysis is representative of at least three different experiments.
  • X4 fusion was measured by a quantitative method while Lee, et al. used microscopic counting of syncytia.
  • HTLV-1 Env Recombinant vaccinia viruses encoding HTLV-1 Env or its transmembrane gp46 subunit were obtained from Dr. H. Shida. gp46 expression was used as a negative control for the specific HTLV-1 Env-mediated cell fusion. This choice was based on the fact that HTLV-1 is a human retrovirus that has a broad range specificity for mammalian cell lines (Sutton, et al.). Target cells expressing ⁇ 32 showed specific reduction in X4 fusion when challenged with the X4 LAV Env. In contrast, the same target cells challenged with HTLV-1 Env-expressing cells showed comparable levels of cell fusion observed with control cells expressing CCR5, MVHA or T7 RNA polymerase (pol3) ( Figure 7).
  • Figure 7 shows the specificity of ⁇ 32-induced inhibition of HIV-1 Env-mediated cell fusion.
  • Target cells (293 or MAGI- CCR5) were coinfected with Ad5pol3 (T7RNA polymerase) and either vLA-2 (pol3+ ⁇ 32) vLA 1 (pol3+CCR5).
  • Figure 8 shows results obtained by the quantitative assay of ⁇ -gal in detergent cell lysates.
  • X4 and R5 fusion showed dramatic reduction as a result of ⁇ 32 expression, whereas no significant effect on X4 fusion was observed with HL60.
  • Control cells expressing recombinant CCR5 did not show significant reduction in X4 fusion.
  • HL60 cells were induced to differentiate into macrophages by RA treatment, the differentiated cells showed the ⁇ 32 effect ( Figure 8B).
  • Figure 8 shows the cell specificity of the ⁇ 32 effect.
  • the effect of ⁇ 32 on X4 fusion is observed with MAGI-CCR5 and 293 cell lines but not with HL60 cell line Figure 8(A).
  • Retinoic acid (RA) treatment of HL60 cell induces them to differentiate and become sensitive to -the ⁇ 32 effect Figure 8(B).
  • Cells were transfected with pCDNA3/CD4 to provide CD4 expression. All cell types were coinfected with pol3 (no ⁇ 32) or vLA 2+pol3 (+ ⁇ 32) and challenged with Hela cells expressing X4 Env (LAV) or R5 (Ba-L) and pT7-lacZ. Cell fusion was measured by ⁇ -gal produced.
  • CD3+CD28 stimulation produces cells that are susceptible for X4 isolates but are resistant to R5 viruses (Carroll, et al.).
  • FIG. 9 shows that PHA+IL-2 activation upregulates CXCR4 and CCR5.
  • PBMCs from seven healthy individuals were stimulated with either PHA Figure 9(A) or PHA+IL-2 and used for cell surface staining of CCR5 and CXCR4 Figure 9(A) or cell fusion assay using one PBMC sample Figure 9(B).
  • CXCR4 levels are variable among individuals confirming published data (Lee, 1999).
  • unstimulated PBMCs respond well in signaling assays using SDF-1 for receptor activation. It appears that the levels of CXCR4 required for signaling assays are different from those required for fusion/infection.
  • the effect of recombinant ⁇ 32 was examined in PBMCs freshly isolated from healthy donors.
  • vLA-2 was used to express recombinant ⁇ 32 in PHA+IL-2 stimulated PBMCs and examined cell surface levels of CXCR4, CD4 and CD44 by FACS analysis. A slight downmodulation effect of CD4 (15-20%) and no effect on CD44 surface expression was observed ( Figure 10A). In contrast, appreciable downmodulation of CCR5 and CXCR4 was observed in the same PBMC population tested ( Figure 10B).
  • Ad5/CCR5 is a replication-defective recombinant virus that does not replicate in primary cells and therefore, it is not expected to result in a robust increase in the levels of CCR5.
  • CCR5 staining of Ad5/CCR5-infected PBMCs showed an increase in CCR5 expression (from 370 to 404, Figure 10B) confirming the delivery of recombinant CCR5 in primary cells.
  • PBMC sample was analyzed by infecting with vLA-1 , vLA2, or Ad5/MVHA and vCB- 21 R (T7-lacZ). Hela cells expressing T7 RNA polymerase and the X4 LAV or Unc Envs were used as effector cells. After mixing the two cell populations and allowing the fusion reaction to incubate for two hours, detergent-treated cell lysates were used to quantitate the amount of ⁇ -gal production as a result of cell fusion. The results demonstrate the ability of recombinant ⁇ 32 to inhibit X4 fusion in these primary cells ( Figure 11 A). PBMCs expressing recombinant CCR5 ( Figure 11 B) or MVHA showed no significant reduction in X4 fusion.
  • PBMC samples isolated from wild type +/+ and homozygous -/- individuals were used to compare cell surface expression of CXCR4.
  • PBMCs were stimulated with PHA+IL-2 for four days then used for FACS staining, cell fusion, and HIV-1 infection assays.
  • the results demonstrate a trend towards lower CXCR4 staining in -/- PBMCs ( Figure 12A).
  • Cell fusion activity with LAV (X4) of -/- cells is always lower th,an that observed with +/+ cells (Figure 12C).
  • Surface expression of CD4 was variable in both +/+ and -/- PBMCs and did not seem lower in -/- compared to +/+ cells ( Figure 12B). The observed CD4 variability is unlikely to influence the data presented here in terms of lower CXCR4 found in -/- individuals since it was observed in both +/+ and -/- samples.
  • Figure 1 1 shows the effect of ⁇ 32 on Env-mediated cell fusion in human PBMCs.
  • the same PBMC samples used for FACS staining in Figure 10B were used in this cell fusion analysis.
  • PBMCs were infected with either vLA-1 (Ad5/CCR5) or vLA-2 (Ad5/ ⁇ 32) at increasing PFUs/cell to ensure expressing recombinant protein in all cells in the monolayer.
  • Infected PBMCs were challenged with Hela cells expressing either Unc or the X4 LAV.
  • PBMCs expressing recombinant CCR5 Figure 1 1 (B) or MVHA Unlike PBMCs expressing recombinant CCR5 Figure 1 1 (B) or MVHA, PBMCs expressing recombinant ⁇ 32 showed a dramatic reduction in X4 fusion Figure 1 1 (A).
  • PBMCs were infected (10 5 cells) with the X4 lab adapted isolate 1MB or the R5 Ba-L and sample supernatants were collected every three days over a 15 days period.
  • the results of this experiment demonstrate that +/+ cells infected with IIIB produced higher amounts of p24 indicating a more efficient productive infection compared to -/- cells ( Figure 13).
  • CXCR4 expression in -/- homozygotes is different from that seen in normal +/+ individuals in terms of surface density and coreceptor function.
  • Figure 12 depicts the results of experiments where PBMCs from individuals with known CCR5 genotype were used. Genotypes are represented by (+) for a wild-type allele and (-) for a ⁇ 32 allele. PBMCs from -/- individuals express lower surface levels of CXCR4, show less cell fusion activity with X4 Env (LAV) and are less susceptible to X4 fusion than +/+ cells. PBMCs from -/- and +/+ individuals were stimulated with PHA+IL-2 for 4 days and used for staining Figure 12(A), cell fusion Figure 12(B), and IIIB infection.
  • LAV X4 Env
  • FIG. 13 shows the infection kinetics of -/- and +/+ PBMCs with HIV-1 IIIB (X4) and Ba-L (R5).
  • PBMCs were stimulated with PHA+IL-2 for 4 days and used in the infectivity assay. Infection was performed in a 96 well plate (10 5 cells/well).
  • Figure 14B shows the identity of the 34 KD band detected with the C-terminal antibodies.
  • the band above the 50 Kd marker band detected in both CCR5 and ⁇ 32 expressing cells could not be CCR5 since it migrates slower (above 50 Kd) than CCR5 that migrates around the 46 Kd marker band ( Figure 14 A).
  • Figure 14 shows the immunoblot analysis of ⁇ 32 and CCR5 proteins expressed in infected 293 cells. Cells were infected with vLA-1 , vLA2, or Ad5 at 10 pfu/cell for 16 hours.
  • FIG. 14(A) shows the monoclonal antisera detect CCR5 but not ⁇ 32 (10% SDS-PAGE);
  • Figure 14(B) shows the C-terminal antibodies detect ⁇ 32 (12% SDS-PAGE) ; and
  • Figure 14(C) shows N-terminal antibodies that detect both CCR5 and ⁇ 32 proteins (10.5% SDS PAGE).
  • ⁇ 32 protein expression in -/- PBMCs was examined using the ⁇ 32-specific antisera.
  • Cell lysates were prepared from unstimulated cells and analyzed the same way described for recombinant ⁇ 32 protein described above.
  • the immunoblot containing three -/- and three +/+ samples was probed with anti- ⁇ 32 antibodies generated against the carboxy terminus of ⁇ 32.
  • the analysis revealed a protein band of different intensity for each -/- individual that corresponds to the same molecular weight band seen with recombinant protein analysis (Figure 15).
  • Approximately, equivalent amounts of protein cell lysates were loaded in each lane. Equal gel loading was verified by staining a similar gel with commassie blue stain.
  • Figure 15 shows the immunodetection of native ⁇ 32 protein expressed in unstimulated PBMCs of three -/- homozygous individuals. The blot was probed with antibodies generated against the carboxy terminus of ⁇ 32 that specifically detect ⁇ 32 protein. Over-exposure did not show any ⁇ 32-related band in +/+ PBMCs. Samples (-/-) 1 , 2 and 3 on this blot correspond to #2, #3, and #6 respectively on Figure 12. Equivalent gel loading was verified by staining a similar gel with commassie blue stain.
  • Samples 1 and 2 in Figure 12(A) correspond to samples 1 &2 in Figure 16.
  • N represents a normal non-genotyped PBMC sample.
  • Southern blot analysis was performed on the gel shown in Figure 16(A) and probed with a 32 P-labeled CCR5/ ⁇ 32-specific DNA Figure 16(C).
  • CCR5 and ⁇ 32-related fragments could have been generated from smaller mRNA species.
  • vLA-1 CCR5
  • vLA-2 vLA-2 ⁇ 32
  • MAGI-CCR5 cells were infected with a recombinant Ad5 encoding T7 RNA polymerase (provided by Frank Graham, McMaster University) at 27 pfu/cell or with a recombinant adenovirus encoding MVHA (Alkhatib, et al.) at 27 pfu/cell and assayed at the same time for PHS 398.
  • Figure 17 shows the effect of recombinant ⁇ 32 protein on X4 infection.
  • MAGI-CCR5 cells were infected with vLA-1 (encoding CCR5) or vLA-2 (encoding ⁇ 32 protein) at increasing viral concentrations (PFU/cell) then infected with the X4 IIIB.
  • HIV-1 infection is quantitated by the amount of ⁇ -galactosidase produced as a result of HIV LTR activation that controls expression of ⁇ -galactosidase.
  • the infection protocol is adapted from Kimpton and Emerman. Values of ⁇ -gal production in cells infected with Ad5pol3 (encoding T7 RNA polymerase) or Ad5/MVHA at 27 pfu/cell were 68 and 60 respectively.
  • Ad5pol3 encoding T7 RNA polymerase
  • Ad5/MVHA at 27 pfu/cell
  • the ability of recombinant ⁇ 32 to provide protection against HIV-1 productive infection in vitro was examined.
  • the experiment was performed by using (vLA-2 ⁇ 32-encoding adenovirus) or, as a negative control, vLA-1 (CCR5-encoding adenovirus) at a moi of 3 pfu/cell. Infected cells were incubated for two days to allow expression of recombinant proteins then infected with either an X4 or a R5 HIV-1. Under these experimental conditions, a pronounced reduction in HIV-1 productive infection was observed (>50% with IIIB Vs 40% with Ba-L; Figure 18).
  • the ⁇ 32 was delivered to PBMCs using Ad5/ ⁇ 32 at 3 pfu/cell and a 50% reduction in infectivity was observed.
  • the efficiency adenovirus vector depends highly on the kind of cells used and a careful standardization of gene delivery into the PBMCs is necessary to perform in order to determine whether full resistance to infection can be obtained with higher doses of ⁇ 32.
  • Figure 18 shows the Phytohemagglutinin-A+IL2-activated Ficoll-purified human PBMCs which were infected with either vLA-1 (Ad5/CCR5) or vLA-2 (Ad5/ ⁇ 32) at 3pfu/cell for each virus for two days then infected with either Ba-L (R5) or IIIB (X4). Infections were performed in a 96-well plate. The virus was absorbed for three hours and cells were washed three times with PBS and maintained in RPMI 1640 supplemented with 10% fetal bovine serum, 100mU of recombinant IL-2 and 10mg/ml PHA.
  • Culture fluid 50 ⁇ l was harvested after cell resuspension every three days and replaced with fresh medium.
  • the amount of p24 antigen in the cell- containing supernatants was measured using an Elisa kit purchased from DuPont.
  • the AZT control infection resulted in p24 values below 0.5 ng/ml.
  • Figure 19 shows the model for the ⁇ 32 effect.
  • ⁇ 32 protein interacts specifically with the major HIV coreceptors interfering with their proper transport to the cell surface.
  • the impairment of coreceptor molecules reduces their chance to associate with CD4 leading to unfavorable stoichiometry for virus entry.
  • the ⁇ 32 effect is concentration-dependent. Higher ⁇ 32 levels can correlate with less coreceptor and reduced virus entry. This concentration effect explains why +/- carriers show partial protective phenotype.
  • Infected -/- cells either expresses low levels of ⁇ 32 or expresses a defective ⁇
  • the data provides evidence for the ⁇ 32 effect on X4 fusion/infection.
  • This experiment examined whether ⁇ 32 protein colocalizes with the major coreceptors and provide evidence for their physical association using protein heterodimerization, co-immunoprecipitation and the yeast 2-hybrid system. These experiments set the stage for experiments focused at analyzing the structural determinants involved in ⁇ 32-coreceptor association. Many integral membrane proteins form noncovalently associated oligomeric complexes that are prerequisites for transport and normal function.
  • the present study is the first to utilize the ⁇ 32 open reading frame containing the frame shift 31 amino acid residues that are not expressed in CCR5.
  • Confocal microscopy was used to detect ⁇ 32-coreceptor complexes using fluorescently labeled molecules.
  • Cells expressing endogenous CCR5 and CXCR4 i.e. MAGI-CCR5 cell line
  • Ad5/ ⁇ 32 was infected with Ad5/ ⁇ 32, examined for the ⁇ 32 effect (downmodulation effect, as in Figure 4) and used to examine whether ⁇ 32 colocalizes with CCR5 or CXCR4.
  • Cells were either fixed or permeablized, and incubated with anti- ⁇ 32 and FITC-labeled secondary antibody.
  • Cell samples were then reacted with either anti-CCR5 or anti-CXCR4 monoclonal antibodies and a phycoerythrin-labeled secondary antibodies. Confocal laser scanning microscopy was performed to detect colocalization of the proteins.
  • Recombinant Ad5 encoding ⁇ 32 or either of the major coreceptors was used to co-infect NIH-3T3 cells.
  • total cell lysates were prepared and immunoprecipitated using the anti-CXCR4 antisera (Feng, et al.) or anti-CCR5 antibodies. Immnuoprecipitation with an antibody followed by western blotting with a different antibody provides a way to determine whether hetero- oligomers form.
  • the immunoprecipitates were fractionated in 10%SDS-PAGE and transferred onto nitrocellulose membranes.
  • the blots were initially probed with anti- ⁇ 32 antibodies to verify the presence of ⁇ 32 protein, then stripped and reprobed with an antibody specific to either CXCR4 or CCR5. Since the ⁇ 32 protein has a lower molecular weight (28-30KD) compared to CXCR4 or CCR5 (45-50KD), it was possible to distinguish the ⁇ 32-CXCR4 or ⁇ 32-CCR5 heterodimers from the CXCR4 or CCR5 homodimers on the immunoblot. Monoclonal antibodies specific to ⁇ 32 protein helped overcome some of the technical difficulties associated with the detection of ⁇ 32-CXCR4 complexes. Co- immunoprecipitation was used as an alternative approaches to confirm the existence of ⁇ 32-coreceptor complexes.
  • Cells co-expressing ⁇ 32 and CCR5 or CXCR4 (recombinant or endogenous) was used for immunopricipitation with anti- ⁇ 32 antibodies.
  • the immune complexes were fractionated and then immunoblotted.
  • Co- precipitated CCR5 or CXCR4 was detected using specific antibodies to either protein.
  • Yeast two-hybrid systems provide a sensitive method for detecting transient protein interactions that are biochemically detectable.
  • the system sensitivity allows quantitative analysis of mutant constructs to be used in the mapping studies.
  • the system has been previously used to analyze CCR5 truncated molecules with wild- type CCR5 (Benkirane, et al.).
  • a MATCHMAKER two-hybrid system purchased from CLONTECH was used to analyze ⁇ 32-co receptor interaction.
  • the cDNA encoding ⁇ 32 ORF was cloned into pGBKT7 (CLONTECH) while the coreceptor cDNA was cloned into pGADT7 (CLONTECH).
  • Yeast AH109 (or Y187) was cotransformed with pGBKT7- ⁇ 32 and pGADT7-coreceptor.
  • the cotransformation mixture was plated on SD/-Leu/-Trp to select for colonies containing both hybrid plasmids.
  • a colony-lift ⁇ -galactosidase assay was used as the system PHS 398.
  • X-gal staining of transformed colonies detects protein interaction. Quantitative analysis of the interaction was performed using 5-bromo-4-chloro-3-indolyl- ⁇ -D- galactopyranoside as a substrate in liquid cultures. The amount of ⁇ -galactosidase made as a result of protein/protein interaction was quantitated using a colorimetric method. Parallel cotransformation controls using pGBKT7- ⁇ 32 and either pGADT7- CXCR2, pGADT7-CD4, or pGADT7-CD44 was performed to control for the background of the system. The preliminary data indicated that ⁇ 32 has no downmodulation effect on CD4, CXCR2 or CD44 ( Figurel O) and therefore, was used as negative controls for coreceptor- ⁇ 32 interactions.
  • ⁇ 32 protein interacts with and specifically inactivates functional expression of other HIV coreceptors.
  • CCR5 and CXCR4 are the major HIV coreceptors
  • CCR3 plays a role in HIV pathogenesis of the brain (He, et al.).
  • Possible contribution of other coreceptors to HIV pathogenesis of other tissues has been recently reported (Lee, et al., Sharron, et al., Faure, et al.).
  • the result of this analysis explains why other coreceptors in -/- individuals are not utilized in the absence of CCR5.
  • the effect of ⁇ 32 on nonhuman CXCR4's were examined to analyze the specificity of ⁇ 32 activity.
  • CD4+ NIH-3T3 cells were coinfected with recombinant adenoviruses encoding ⁇ 32 and the appropriate coreceptor (or a chemokine receptor) and Ad5pol3 (T7 RNA Polymerase). Effector Hela cells was coinfected with vCB-21 R (T7lacZ) and one of the HIV-1 Envs-expressing vaccinia vectors.
  • the recombinant adenovirus expressing ⁇ 32 protein was used as an antigen to immunize the mice. This provides a method to deliver ⁇ 32 in its native conformation. These experiments take place in the animal colony of Indiana University medical center.
  • the generated MAbs was used as reagents to detect ⁇ 32, and to map its functional domain(s) using a panel of point mutants. Since Mabs were generated using the vLA-2 that delivers native ⁇ 32, generated antibodies had different specificities that helped map critical epitopes in ⁇ 32. This was accomplished by examining the ability of ⁇ 32 variants (chimeras and point mutants) to dbwnmodulate coreceptors and be recognized by a particular Mab.
  • ⁇ 32 protein impairs the formation of CD4-Coreceptor complexes by reducing coreceptor availability.
  • Coreceptors compete for association with CD4 (Lee, et al.) ⁇ 32 protein reduces this interaction by associating with the coreceptor.
  • CD4-coreceptor complexes exist without the presence gp120 and by using confocal microscopy the colocalization of such complexes has been demonstrated
  • CD4-CCR5 complexes are stronger than CD4-CXCR4 complexes. It is not known however, whether CD4- coreceptor association occurs cotranslationaly or at the cell surface.
  • Hela cells were used in this analysis because it expresses endogenous CXCR4.
  • a CD4-encoding vaccinia virus using different doses of the virus delivers increasing levels of CD4. These expression levels were established by infecting Hela cells with 1 pfu/cell (low), 5 pfu/cell (medium) or 20pfu/cell (High). Cells were verified for CD4 expression by quantitative FACS analysis.
  • the ⁇ 32 protein was introduced into these cells by Ad5/ ⁇ 32 at an inhibitory dose (10-20pfu/cell) and challenged with X4 or R5 Env-expressing cells. The effect of high CD4 levels delivered by the vaccinia vector was assayed by quantitating X4 and R5 fusion compared to control virus-infected cells.
  • CD4 associates with coreceptor cotranslationally, then it is possible to colocalize the CD4-co receptor ⁇ 32 by confocal microscopy.
  • a similar approach was taken to study the effect of CCR5 and CXCR4 concentrations on ⁇ 32 effect in cells expressing limited amounts of CD4. This analysis determines whether the ratio of pre-existing CD4-CCR5 to CD4-CXCR4 complexes influences ⁇ 32 effect.
  • Cell lines such as MAGI-CCR5 were suitable for this kind of analysis since it contains all molecules involved in the process.
  • the levels of CD4 expressed by vaccinia virus vectors are very high and may not be relevant to the in vivo situation. Additionally, high multiplicity infection can change membrane structures leading to high background levels.
  • cell lines expressing different levels of CD4 were used and assayed for its effect on intracellular ⁇ 32.
  • CCR2 shares more than 75% homology with CCR5 Alkhatib, et al., it was not downmodulated by ⁇ 32 suggesting a possible specific interaction that involves at least, part of the non-homologous region.
  • ⁇ 32 expression has no significant effect on surface levels of CD4 confirming that the observed reduction in X4 fusion/infection is not due to an effect on CD4.
  • CCR5/CCR2 hybrid constructs and CCR5 point mutants were also utilized to analyze the region of CCR5 that is likely to interact with ⁇ 32.
  • CXCR4 point mutants were utilized to determine a potential region of CXCR4 that interacts with ⁇ 32.
  • the unique carboxy terminal 31 amino acids in the ⁇ 32 protein create new functional domain(s) that can be specific for interaction with coreceptors.
  • a direct way to determine whether the carboxy terminal region of ⁇ 32 is critical for its activity is to construct chimeric molecules where this unique ⁇ 32- specific COOH tail or portion of it is grafted onto CCR5 that lacks its carboxy tail.
  • CCR5 lacking the carboxy terminal cytoplasmic tail is expressed at the cell surface and is functional as a coreceptor (Alkhatib, et al.).
  • Co-expression of tail-less CCR5 and wild type CCR5 or CXCR4 had no effect on R5 or X4 fusion, which is in agreement with published data using a similar construct (Benkirane, et al.).
  • mutants can be designed to include the shared homologous region with CCR5.
  • CXCR4 and CCR5 share only 30% homology (including conserved amino acid changes), it is important to examine whether a common region of the principal coreceptors CXCR4 and CCR5 is responsible for interacting with ⁇ 32. Knowledge of this common region provides insight into the molecular basis of coreceptor activity and the design of new drugs that can have a broader antiviral effect.
  • a direct method to map important regions of coreceptor involved in interaction with ⁇ 32 can be to construct chimeric molecules between CXCR4 or CCR5 with homologs that do not function as coreceptors or show a different coreceptor function in terms of Env specificity (i.e. CCR2 works only with 89.6 but not with R5 Envs).
  • the hybrid molecules are transfected into 293 cells followed by infection with Ad5/ ⁇ 32 and FACS analysis to determine the hybrid molecules that lost the ability to associate with ⁇ 32.
  • the loss of function is characterized by the ability of the hybrid molecule to escape the effect of ⁇ 32 indicative by its efficient cell surface expression.
  • Obtaining functional mutants is also useful to verify and confirm the interactive region.
  • CXCR4 point mutants provided by Chris Broder (Chabot, et al.)
  • CCR5 point mutants Alkhatib, et al.
  • Most CXCR4 point mutants are expressed at the cell surface and therefore, are assayed for the ⁇ 32 effect by FACS analysis.
  • Antibodies to ⁇ 32 are now available and are being used to specifically detect ⁇ 32 protein in Western blots and immunoprecipitation. One first determines whether ⁇ 32 physically associates with the major coreceptors. The biological analyses of chimeric mutants as well as the heterodimer formation experiments can initially use the polyclonal antibodies for detecting the mutant protein. Once the Mabs are made they are used with ⁇ 32 chimeras and point mutants to map the critical determinants of this protein.
  • CCR5/ ⁇ 32 heterozygosity confers a different degree of protection against HIV-1 in PBLs and MDMs, depending on the ratio of wild-type and mutant ⁇ 32 mRNA in the two cell types.
  • the investigators analyzed expression levels of wild type and mutant ⁇ 32 mRNA species by competitive RT-PCR and demonstrated that depending on the cell type, ⁇ 32 heterozygosity confers a different degree of protection against HIV-1 infection.
  • Antibodies raised against the COOH-terminus of ⁇ 32 to analyze its expression in PBMCs of -/- and +/- individuals were used.
  • a number of -/-, +/-, and +/+ PBMCs are used.
  • Cell lysates of unstimulated PBMCs, or PHA-stimulated PBMCs from homozygotes and heterozygotes are prepared and analyzed for expression of endogenous ⁇ 32 protein by immunoblotting and immunoprecipitations as presented in the preliminary data section. Quantitative protein analysis are performed to determine whether there are differences in ⁇ 32 protein levels in -/- versus +/- individuals that can explain the partial, incomplete protective phenotype in heterozygotes. Similar analysis are performed on the infected -/- PBMC sample to determine whether ⁇ 32 protein is expressed in these cells.
  • PBMCs from one infected ⁇ 32 homozygote is not representative of all 8 homozygotes identified so far (Michael, et al.). Unfortunately, some of these individuals are dead. and it is possible to examine their samples.
  • PBMCs from -/- individuals provide an in vivo system where the effect of ⁇ 32 on endogenous coreceptors can be assayed in their native environment.
  • ⁇ 32 protein is expressed and can be detected in -/-PBMCs ( Figure 15). It is important to examine ⁇ 32-CXCR4 interaction in -/- PBMCs to provide an in vivo evidence for this molecular association. As mentioned earlier, the association of ⁇ 32 and CXCR4 is relevant to pathogenesis.
  • Pulse chase experiments are performed to compare coreceptor protein synthesis in -/- and +/+ PBMCs to directly examine biosynthesis and stability of coreceptor proteins made in these two cell populations. Unstimulated as well as PHA+IL2 stimulated -/- and +/+ PBMCs are used for labeling with 35 S-methionine.
  • Activated cells express higher levels of endogenous proteins and therefore can be useful in tracing coreceptor proteins in pulse-chase experiments.
  • the protein analysis shown in Figure 15 indicated the detection of ⁇ 32 protein in unstimulated cells.
  • cell lysates are prepared and used in co-immunoprecipitation assays.
  • the effect on stability of CXCR4 in -/- versus +/+ PBMCs is examined in pulse chase experiments followed by immunoprecipitation of CXCR4 with monospecific antibodies (Feng, et al.).
  • the experiment is focused on developing a cellular model system to examine other factors that can contribute to resistance to HIV-1. Identification of these factors opens new avenues in this field and has important implications for understanding the molecular basis of resistance to HIV-1.
  • the human myeloid HL-60 cell line can be differentiated into macrophages upon treatment with RA.
  • RA treatment Upon RA treatment, these cells differentiate to acquire a macrophage phenotype and become fusogenic with R5 tropic HIV-1 (Alkhatib, et al.). Since the ⁇ 32 effect is observed in HL60 differentiated cells, then it suggests expression of another cellular protein that can be critical for the ⁇ 32 effect.
  • ⁇ 32 can downmodulate CXCR4 in RA-treated HL60 cells using untreated cells as a negative control.
  • Subtraction cDNA library is made using undifferentiated and RA-differentiated cells as a source of RNA.
  • the induced molecule(s) is required for ⁇ 32 then it can interact with it.
  • a direct method to examine and isolate the interacting protein is using the yeast two-hybrid screening system. Once the cDNA is/are isolated, it is transfected into untreated HL60 cells to examine if it can transfer the ⁇ 32 effect.
  • the cDNA clone is sequenced and its identity and homology with other proteins are analyzed.
  • the induced molecules in RA-treated HL60 cells cannot interact with ⁇ 32 but can interact with CXCR4.
  • Another two-hybrid screening is performed using CXCR4 as the bait protein. If these screening methods failed to identify the induced molecule, a functional cDNA screening method is used to isolate the cDNA clone. This method is based on the transfer of ⁇ 32 function to untreated HL60 cells. Briefly, the cDNA library under T7 promoter is transfected into HL60 cells, followed by infection with Ad5pol3 (T7 RNA pol) and Ad5/ ⁇ 32. Reduction in X4 fusion indicates transfer of function. The cDNA library is then subfractionated and fractions are used to repeat the screening until a single clone is identified.
  • Cell lines transformed with ⁇ 32 provide a cellular model for the analysis of ⁇ 32 activity and its contribution to resistance to HIV-1. Creation of this cell line is critical to analyze ⁇ 32-CXCR4 complex formation in cells synthesizing endogenous levels of ⁇ 32 and CXCR4 proteins. This is a ⁇ 32-CXCR4 interaction without the presence of endogenous CCR5. This allows studying the effect of introduction of exogenous CCR5 in order to determine whether the major coreceptors compete for complexing with ⁇ 32. The analysis of ⁇ 32 has relied on Ad5 vectors to express recombinant ⁇ 32 in cell lines as well as primary cells. In all experiments the ⁇ 32 gene was introduced into cells expressing endogenous CXCR4 and/CCR5.
  • FIG. 17 and 18 The preliminary data shown in Figures 17 and 18 indicated that cell lines as well as PBMCs expressing recombinant ⁇ 32 showed reduced susceptibility to HIV-1 infection. It is of basic interest to examine ⁇ 32 effect in a CD4+ T-cell line that expresses endogenous levels of ⁇ 32 protein.
  • Jurkat cells are CD4+CXCR4+ and endogenous expression of ⁇ 32 directly address its effect on endogenous CXCR4 expression and susceptibility to infection. This allows detailed analysis of the intracellular distribution and localization of ⁇ 32 protein and its possible interaction with CXCR4.
  • the choice of Jurkat cell line is based on the fact that it is CD4+CXCR4+ and it shows ⁇ 32 effect when infected with Ad5/ ⁇ 32.
  • Standard procedures are used as previously described to create a Jurkat- CCR5 cell line (Alkhatib, et al.). Positive clones are identified by PCR on cellular genomic DNA. Clones are selected on the basis of the ⁇ 32 made using RT-PCR as a method of quantification. Clones with low, medium, and high levels of ⁇ 32 mRNA expression are selected and expanded. Antibodies to ⁇ 32 can finally be used to identify the clones expressing low, medium, or high levels of the ⁇ 32 protein. Once clones of ⁇ 32 cell lines are purified they can be tested in infectivity assays to determine the percent reduction in HIV-1 fusion/infection assays. The selected clones expressing different levels of ⁇ 32 are extremely useful in determining whether a dose-effect correlation can exist in vivo.
  • Example 2 The Yeast Two-hybrid system was used to provide evidence for CXCR4 interaction with the Delta-32 protein.
  • GAL4-based Matchmaker two-hybrid system 3
  • the system provides a transcriptional assay for detecting protein interactions in vivo in yeast.
  • Delta-32 gene was expressed as a fusion to the GAL4 DNA-binding domain (DNA-BD), while CCR5 or CXCR4 was expressed as a fusion to the GAL4 activation domain (AD).
  • DNA-BD fusion vector, pGBKT7 and AD fusion vector pGADT7 were used for high level expression.
  • Delta-32 (cloned in pGBKT7) and CCR5/CXCR4 (cloned in pGADT7) inserts were expressed as GAL4 fusions with c- Myc and hemagglutinin (HA) epitope tags, respectively.
  • the transcription and translation of epitope-tagged fusion proteins in vitro was driven by T-7 promoter, which is at the downstream of the GAL4 coding sequence.
  • Cotransformation was carried out using both the bait -Delta-32 and AD fusion vector-CCR5/CXCR4 in yeast strain AH 109, which is gal4 " and gal80 " prevents interface of native regulatory proteins with the regulatory elements in the two-hybrid system.
  • pGBT7-53 and pGADT7-T encode fusions between the GAL4 DNA-BD and murine p53 and SV40 large T-antigen, respectively.
  • p53 and large T-antigen interact in a yeast two-hybrid assay and was used as positive control.
  • PGBKT-7-Lam encodes a fusion of the DNA-BD with human lamin C and provides a control for a fortuitous interaction between an unrelated protein and the pGADT7-T and was used as a negative control.
  • Exp.#1 is a negative control.
  • Exp.#2 is a positive control for interaction of two well-known proteins.
  • Exp. #3, 4, and 5 are all controls to measure the background of the system.
  • Exp.#6 and 7 represent confirmed results of ⁇ -32 protein interaction with not only CCR5 but also CXCR4.
  • Interaction of ⁇ -32 with CXCR4 is a novel finding that has never been described in the field of AIDS. This finding is significant because CXCR4 is linked to disease progression and represent an important coreceptor that the majority of HIV-1 isolates utilize during the symptomatic stage of AIDS.
  • Example 3 Example 3:
  • HIV-1 Human immunodeficiency virus type 1
  • CD4 and a coreceptor, principally the CCR5 and/or CXCR4 chemokine receptors, for entry into host cells.
  • CCR5 The central role of CCR5 in HIV-1 transmission and pathogenesis has been highlighted by the epidemiological and genetic identification of powerful disease-modifying effects of the naturally occurring CCR5 ⁇ -32 (D32) allele, a 32- base pair deletion encoding a truncated and non-cell surface expressed version of CCR5. Relative to the general population, D32/D32 homozygotes are rarely found among HIV+ individuals but are significantly more common among repeatedly exposed/uninfected (EU) individuals.
  • EU repeatedly exposed/uninfected
  • D32 delta-32
  • ORF open reading frame
  • HIV-1 Human immunodeficiency virus type 1
  • CD4 and a coreceptor, principally the CCR5 and/or CXCR4 chemokine receptors, for entry into host cells.
  • the central role of CCR5 in HIV-1 transmission and pathogenesis has been high-lighted by the epidemiological and genetic identification of powerful disease modifying effects of the naturally occurring CCR5 ⁇ 32 allele, a 32 base pair deletion encoding a truncated and non-cell surface expressed version of the coreceptor.
  • ⁇ 32 Delta-32
  • ORF open reading frame
  • ⁇ 32 protein expression can specifically reduce the expression of endogenous CCR5 or CXCR4 resulting in the inhibition of virus entry and infection by R5 and X4 HIV-1 isolates. This effect was not observed in cells expressing recombinant CCR5 or other non-relevant proteins such as measles virus hemaglutinin (MVHA) or structural adenovirus proteins.
  • MVHA measles virus hemaglutinin
  • ⁇ 32 protein down-regulates the major coreceptors resulting in an unfavorable stoichiometry of the molecules involved in viral entry.
  • ⁇ 32-specific antibodies we have shown that ⁇ 32 is expressed in ⁇ 32/ ⁇ 32 PBMCs.
  • HIV-1 human immunodeficiency virus type 1
  • CD4 human immunodeficiency virus type 1
  • CXCR4 the major coreceptors used by most R5 and X4 HIV-1 isolates.
  • the importance of chemokine receptors in HIV-1 transmission is highlighted by the finding that individuals homozygous for a 32-base pair deletion in CCR5 ( ⁇ 32/ ⁇ 32) are resistant to HIV-1 infection.
  • the defective coreceptor gene encodes a prematurely terminated protein (Fig.lA) that is not detected at the cell surface and therefore is not functional as a fusion coreceptor.
  • ⁇ 32 homozygous as -/-
  • ⁇ 32 heterozygous as +/-
  • Genotypic analysis of this mutation and its distribution revealed that ⁇ 32 has a high allele frequency among Caucasians but was absent in African or Asian populations.
  • the mutant allele is not associated with any obvious phenotype in uninfected homozygous individuals.
  • Heterozygotes (+/-) are not protected against infection, but once they become infected, have a slower progression to AIDS, indicating that partial resistance can occur in the presence of a single copy of the mutant CCR5 gene.
  • the frameshift ⁇ 32 mutation introduces 31 new amino acid residues at the carboxy terminus of ⁇ 32 that are not present in CCR5 (Fig. 1A&C).
  • the CCR5 ⁇ 32 protein was expressed and analyzed in 293 cells infected with a recombinant adenovirus Ad5/ ⁇ 32 ( Figure 1 B) (which also express green fluorescent protein) using a 31 -amino acids custom peptide antibody generated against the carboxy terminus of ⁇ 32-ORF ( Figure 1C).
  • the ⁇ 32 protein was found to be abundantly expressed in 293 cells infected with Ad5/ ⁇ 32 and showed a protein band at around 28-30 kDa ( Figure 2A &B)).
  • the ⁇ 32 protein was also detected using anti-CCR5 antiserum directed against the common N-terminus of CCR5 and CCR5 ⁇ 32.
  • immunostaining of ⁇ 32 protein indicated that it is expressed intracellularly towards the inner side of the plasma membrane ( Figure 2C).
  • PBMCs freshly isolated from healthy donors.
  • PBMCs were stimulated with PHA+IL-2 for 3 days then used for FACS staining and cell fusion.
  • the PBMCs were infected with Ad5/ ⁇ 32 and examined for ⁇ 32 effect on cell surface levels of CCR5, CXCR4, CD4, CD25, CD44, and CXCR2 by FACS analysis.
  • Ad ⁇ MVHA, or wild type Ad5 vector was included infected with Ad5/CCR5.
  • the degree of fusion inhibition was proportional to the amount of expressed ⁇ 32 protein made in the infected PBMCs ( Figure 3, protein blot).
  • the gradual increase in the inhibitory effect was proportional to the increasing intensity of ⁇ 32 protein band.
  • we found that the levels of ⁇ 32 protein obtained by our AD ⁇ / ⁇ 32 could not be obtained by simple DNA transfection of 293 cells (data not shown) which may explain the inability of Venketesan, et al. (Bleul, et al.) to observe the ⁇ 32 effect.
  • RNA expression of ⁇ 32 was verified by RT-PCR analysis ( Figure 6A).
  • the immunoblot analysis revealed a protein band (Figure 6B&C) that corresponds to the same molecular weight band seen with recombinant protein analysis ( Figure 2). These protein bands were not obtained with the preimmune serum. The identity of the band that appears above the 34 Kd marker band is not known at present, however, since it is expressed in normal CCR ⁇ individuals, it could represent a cross- reactive cellular protein.
  • Our analysis also revealed that the ⁇ 32 protein band detected in the protected (-/-) individuals was absent in an infected (-/-) individual ( Figure 6C) suggesting a critical role for the ⁇ 32 ORF in resistance to HIV.
  • Recombinant ⁇ 32 conferred a broad protective effect against X4 and R ⁇ HIV- 1 infection.
  • ⁇ 32 protein has a lower molecular weight (28-30kDa) compared to CXCR4/CCR ⁇ (45- 50KD), it will be possible to distinguish the ⁇ 32-CXCR4 or ⁇ 32-CCR ⁇ heterodimers from the CXCR4 or CCR ⁇ homodimers on the immunoblot.
  • Anti-c-myc or anti-HA antibodies will also be used for immunoprecipitating ⁇ 32 with CXCR4 or CCR5 in yeast two hybrid system as ⁇ 32 and the coreceptors are expressed as fusion proteins of c-myc and HA.
  • polyclonal antibodies to specifically detect recombinant and native ⁇ 32 protein.
  • Monoclonal antibodies to ⁇ 32 protein may help overcome some of the technical difficulties associated with the detection of ⁇ 32- CXCR4 complexes.
  • Co-immunoprecipitation will be used as an alternative approaches to confirm the existence of ⁇ 32-coreceptor complexes.
  • Cells co- expressing ⁇ 32 and CCR ⁇ or CXCR4 (recombinant or endogenous) will be used for immunoprecipitation with anti- ⁇ 32 antibodies.
  • the immune complexes will be fractionated and then immunoblotted.
  • Co-precipitated CCR ⁇ or CXCR4 will be detected using specific antibodies to either protein.
  • the recombinant adenovirus Ad ⁇ / ⁇ 32 is used as an antigen to immunize the mice. These experiments take place in the animal colony of Indiana University medical center. This will deliver the ⁇ 32 protein in its native conformation providing an opportunity to generate Mabs to native ⁇ 32 epitopes. Examining the ability of ⁇ 32 variants (chimeras and point mutants) to downmodulate coreceptors and be recognized by a particular Mab will help generate a functional map of ⁇ 32. By generating a series of Mabs and continuing these downmodulation experiments we will be able to generate a functional map of ⁇ 32.
  • Subcellular localization For intracellular co-localization studies, paraformaldehyde-fixed cells will be washed once with HBSS/BSA and then incubated in HBSS/BSA containing 0.0 ⁇ % saponin for 30 min at room temperature.
  • ER endoplasmic reticulum
  • PDI protein disulfide isomerase
  • a direct way to determine whether the carboxy-terminal region of ⁇ 32 is critical for its activity will be to construct chimeric molecules where this unique ⁇ 32- specific COOH tail or portion of it is grafted onto CCR ⁇ that lacks its carboxy tail (gain of ⁇ 32 function).
  • CCR ⁇ lacking the carboxy terminal cytoplasmic tail is expressed at the cell surface and is functional as a coreceptor. If the biological activity of ⁇ 32 is contained within the last 31 amino acids at its carboxy terminus, then adding this region to a tail-less CCR ⁇ is expected to result in the loss of its ability to reach the cell surface. This analysis will be extended to include other truncation of this ⁇ 32 fragment in order to map its interactive domain.
  • the ⁇ 32 may be used as a therapeutic approach to induce resistance to HIV-1 infection.
  • Gene delivery of the ⁇ 32 to stem cells may provide the opportunity of producing progeny cells that resist HIV-1 infection.
  • the ⁇ 32 protein is a naturally expressed molecule and individuals.- carrying this mutation do not show any obvious hematopoietic defects or any other immunological disorders.
  • CCR3 and CCR ⁇ are co-receptors for HIV-1 infection of microglia," Nature, 38 ⁇ (6617) 64 ⁇ -9 (1997).
  • CCR5 coreceptor usage of non-syncytium-inducing primary HIV-1 is independent of phylogenetically distinct global HIV-1 isolates: delineation of consensus motif in the V3 domain that predicts CCR-5 usage," Virology, 240(1), 83-92 (1998).
  • Alkhatib, G. et al. "High-level eucaryotic in vivo expression of biologically active measles virus hemagglutinin by using an adenovirus type 5 helper-free vector system," J. Virol., 62, 2718-2727 (1988).
  • Alkhatib, G., et al. "Expression of bicistronic measles virus P/C mRNA by using hybrid adenoviruses: levels of C protein synthesized in vivo are unaffected by the presence or absence of the upstream P initiator codon," Journal of Virology, 62(1 1 ), 4059-69 (1988).
  • HIV-1 Co-receptor activity share common structural determinants: Critical residues in the third extracellular loop support HIV-1 fusion," Journal of Biological Chemistry, 272(32), 19771 -6 (1997).

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Abstract

La présente invention se rapporte à une méthode de protection de personnes contre la contamination par le VIH, qui consiste à administrer soit un vecteur soit un vaccin contenant une séquence codant la mutation Δ32. L'invention se rapporte également à une méthode permettant de réduire la quantité des co-récepteurs présents à la surface des cellules et consistant à administrer un composé incluant une séquence codant la mutation Δ32 dans un excipient pharmaceutiquement acceptable. L'invention se rapporte également à un composé permettant de réduire la quantité des co-récepteurs du VIH présents à la surface des cellules, ce composé comportant une séquence codant la mutation Δ32 dans un excipient pharmaceutiquement acceptable. L'invention se rapporte également à un vecteur contenant une séquence codant la mutation Δ32, ainsi qu'à une analyse permettant de tester l'efficacité d'un traitement contre le VIH, ladite analyse consistant à utiliser un détecteur permettant de déceler la présence de la mutation Δ32 dans des cellules.
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